This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Considerable effort has been made to develop drugs against mosquito-borne flaviviruses, such as dengue virus (DENV), West Nile virus (WNV) and Japanese encephalitis virus (JEV). However, to date no such antiviral drugs are available. Growing evidence suggests that a particular area of the so-called stem region of the outer coating, or envelope protein, of these viruses is involved in virus entry and virus assembly. The stem contains several highly conserved amino acids among different flaviviruses. We conjecture that these highly conserved amino acids play important roles in virus entry and virus assembly. The objective of this study is to analyze the stem region of DENV and other medically important flaviviruses for novel targets that would lead to the development of antiviral drugs. In Aim 1, we will investigate the role of the highly conserved amino acids on the entry and assembly of DENV serotype 4. In Aim 2, we will investigate the mechanisms of impairment on the entry and assembly of DENV serotype 4. And in Aim 3, we will investigate the role of the highly conserved amino acids on the entry and assembly of other DENV serotypes, as well as WNV and JEV. Characterization of the roles of the highly conserved amino acids on virus assembly and entry would lead to new insights about the molecular mechanisms of flavivirus replication. In turn, this new knowledge will lead to the development of new antiviral drugs to reduce the human suffering and deaths associated with diseases caused by DENV and other flaviviruses.